POLYMERASE CHAIN-REACTION AMPLIFICATION AND SEQUENCE-ANALYSIS OF HUMAN MUTANT ADENINE PHOSPHORIBOSYLTRANSFERASE GENES - THE NATURE AND FREQUENCY OF ERRORS CAUSED BY TAQ DNA-POLYMERASE

被引：39

作者：

CHEN, J

SAHOTA, A

STAMBROOK, PJ

TISCHFIELD, JA

机构：

[1] INDIANA UNIV,SCH MED,DEPT MED GENET,975 W WALNUT ST,INDIANAPOLIS,IN 46202

[2] UNIV CINCINNATI,COLL MED,DEPT ANAT & CELL BIOL,CINCINNATI,OH 45267

来源：

MUTATION RESEARCH | 1991年 / 249卷 / 01期

关键词：

APRT SEQUENCE; M13 AND DIRECT SEQUENCING; MOLECULAR CLONING; POLYMORPHISM; NUCLEOTIDE MISINCORPORATIONS;

D O I：

10.1016/0027-5107(91)90143-C

中图分类号：

Q3 [遗传学];

学科分类号：

071007 ; 090102 ;

摘要：

Using the polymerase chain reaction (PCR) with Taq DNA polymerase, we have amplified a 2.4-kb fragment of genomic DNA containing the adenine phosphoribosyltransferase (APRT) gene from patients with APRT deficiency. Several clones from each patient were sequenced after subcloning the PCR product into M13mp18. Selected regions of the amplified fragment were also sequenced directly. This enabled us to distinguish PCR-induced errors from endogenous mutations and polymorphisms in each clone. 44 PCR errors were found in a total of 57.94 kb of DNA sequenced from 25 clones from 7 patients. All the errors were due to the PCR process and not to subcloning, as shown by sequence analysis of 5 APRT-positive clones isolated from a phage genomic library.

引用

页码：169 / 176

页数：8

共 19 条

[1] ENZYMATIC AMPLIFICATION OF EXOGENOUS AND ENDOGENOUS RETROVIRAL SEQUENCES FROM DNA OF PATIENTS WITH TROPICAL SPASTIC PARAPARESIS [J].